PROJECT SUMMARY/ABSTRACT Purpose of the project. This application focuses on the use of imaging to better understand, reverse, and monitor immune suppression and metabolism in murine models of aggressive/metastatic breast cancer. High LDH-A and monocarboxylate transporters 1 and 4 (MCT-1 and MCT-4) have been linked to poor prognosis, and greater metastatic potential. Based on clinical analyses of GEO and MSKCC?s cBio Portal: i) tumors with increased expression of genes involved in lactate metabolism are more aggressive and have poor survival, and ii) there is an inverse correlation between the expression of glycolysis genes and immune-related genes. These data support our hypothesis: that high rates of tumor glycolysis leads to a lactic acid-rich tumor microenvironment (TME) with the exclusion of T cells, resulting in more aggressive tumors with a propensity to form metastases. We further hypothesize that reversal of the T cell exclusion with metabolic inhibitors will render tumors that are resistant to immune modulation with checkpoint blockade (CTLA-4, PD-1) to be responsive to the treatment, due to repopulation of T cells within the tumor microenvironment. Here, we propose to use multimodal imaging to explore the mechanisms by which metabolic and immune modulation therapy reverse the restriction and inactivation of cytotoxic T cells in clinically relevant murine models of aggressive breast cancer. We plan to: 1) characterize in vivo changes in tumor lactate and T cell infiltration during LDH-A and MCT-1/4 inhibition; 2) evaluate the responses to adjuvant and neo-adjuvant single and combination therapy (metabolic inhibition and ?checkpoint? blockade in vivo); 3) validate the imaging results by assessing correlations between glycolytic biomarkers and T cell infiltration using ex vivo immunofluorescence (IF), immunohistochemistry (IHC), and fluorescence-assisted cell sorting (FACS); and 4) assess the potential for clinical translation. The translational goal is two-fold: 1) to induce regression in primary and metastatic breast cancer, by increasing tumor penetration and effector function of cytotoxic T cells, and 2) to monitor treatment response by non-invasive imaging. Experimental Strategy. We have established several murine models of aggressive breast cancer in immune competent host animals. In Aim 1, in vitro and in vivo studies will define how tumor-cell metabolism affects T cell function, and identify a ?therapeutic window? for optimizing the magnitude and timing of T cell repopulation of aggressive murine breast tumors following LDH and MCT pharmacologic inhibition. In Aim 2, metabolic and immune-PET imaging will monitor how changes in the TME (induced by anti-LDH and anti-MCT treatment) affect T cell infiltration and function. Based on Aim 1 and 2 studies, an ?optimized? lactate inhibition treatment strategy (established in Aims 1 and 2) will be combined with immune checkpoint blockade (anti-PD1 and anti- CTLA4) and assessed in Aim 3. Imaging will be used to quantitate tumor lactate, the conversion of hyperpolarized 13C-pyruvate to lactate, glycolysis (reflected in FDG uptake) and the trafficking of CD8+ T cells.